In other cases, the protoplasts were diluted into molten
soft agar at 45 1C containing either LB medium and
0.5 M sucrose or M9 medium, 0.5 M sucrose, and either
Leu (100 mg/ml) or Arg (100 mg/ml), depending on the
nutritional requirements of the auxotrophic strains used,
and then poured onto soft agar plates of the same
composition.

Overview

Replace this sentence with a brief description of the protocol and its goal.

Materials

List reagents, supplies and equipment necessary to perform the protocol here. For those materials which have their own OWW pages, link to that page. Alternatively, links to the suppliers' page on that material are also appropriate.

supply 1 (i.e. tubes of a certain size? spreaders?)

reagent 1

X μL reagent 2

component A (reagent 2 is made up of multiple components)

component B

equipment 1

equipment 2

Procedure

Strip outer membrane

Pellet cells

Pour off supernatant

Resuspend in Tris-HCl

Add EDTA slowly

Shake

Pellet and rinse

Permeabilize cell wall

Pellet, rinse with SMM, resuspend in SMM + lysozyme

Shake and incubate

No current step to stop lysozyme

Should have protoplasts now

Optional: Fuse protoplasts

Mix aliquots of protoplasts of each parent strain

Add PEG

Step 3

Regenerate protoplasts

Step 1

Step 2

Step 2 has some additional information that goes with it. i.e. Keep at 4°C.

Step 3

Step 3 has multiple sub-steps within it.

Enumerate each of those.

Notes

List troubleshooting tips here.

You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.

Anecdotal observations that might be of use to others can also be posted here.

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